Elevated interleukin-8 expression by skin fibroblasts as a potential contributor to pain in women with Fabry disease

Fabry disease (FD) is a lysosomal storage disorder of X-linked inheritance. Mutations in the α-galactosidase A gene lead to cellular globotriaosylceramide (Gb3) depositions and triggerable acral burning pain in both sexes as an early FD symptom of unknown pathophysiology. We aimed at elucidating the link between skin cells and nociceptor sensitization contributing to FD pain in a sex-associated manner. We used cultured keratinocytes and fibroblasts of 27 adult FD patients and 20 healthy controls. Epidermal keratinocytes and dermal fibroblasts were cultured and immunoreacted to evaluate Gb3 load. Gene expression analysis of pain-related ion channels and pro-inflammatory cytokines was performed in dermal fibroblasts. We further investigated electrophysiological properties of induced pluripotent stem cell (iPSC) derived sensory-like neurons of a man with FD and a healthy man and incubated the cells with interleukin 8 (IL-8) or fibroblast supernatant as an in vitro model system. Keratinocytes displayed no intracellular, but membrane-bound Gb3 deposits. In contrast, fibroblasts showed intracellular Gb3 and revealed higher gene expression of potassium intermediate/small conductance calcium-activated potassium channel 3.1 (KCa 3.1, KCNN4) in both, men and women with FD compared to controls. Additionally, cytokine expression analysis showed increased IL-8 RNA levels only in female FD fibroblasts. Patch-clamp studies revealed reduced rheobase currents for both iPSC neuron cell lines incubated with IL-8 or fibroblast supernatant of women with FD. We conclude that Gb3 deposition in female FD patient skin fibroblasts may lead to increased KCa3.1 activity and IL-8 secretion. This may result in cutaneous nociceptor sensitization as a potential mechanism contributing to a sex-associated FD pain phenotype.


Introduction
The inherited disorder Fabry disease (FD) is caused by several hundred mutations in the gene encoding the enzyme α-galactosidase A (GLA) [1].Reduction or absence of GLA activity results in a multi-organ disease with limited life expectancy and lysosomal depositions of globotriaosylceramide (Gb3) [2].Although FD is X-linked, both sexes may reach all levels of disease severity.One of the earliest symptoms of FD is episodic acral burning pain [3] which is typically triggered by heat, inflammation or physical activity [4] and is mostly attributed to small nerve fiber impairment.Varying FD pain phenotypes reduce patients'health-related quality of life, while the pathophysiology remains unknown.Gb3 deposits in dorsal root ganglion (DRG) neurons of FD patients [5] and FD animal models [6][7][8] provide hints towards a link between neuronal sphingolipid accumulation and FD-associated pain.In contrast, the recently reported association of Gb3 accumulation in fibroblasts and FD-associated pain in men with FD [9] suggests another potential mechanism contributing to pain.Skin fibroblasts and keratinocytes are key players in the processing of chemical and thermal stimuli and cutaneous nociception [10][11][12][13].Additionally, dermal fibroblasts also express pain-associated ion channels [14][15][16] and are linked with pro-inflammatory cytokine release in patients with small fiber neuropathy [17].Further research underscores the role of fibroblast control of nociception in animal and in vitro studies [18][19][20].Mechanisms of potential cross-talk of both pathways and sex-specificity remain elusive.We aimed to unravel Gb3-dependent cutaneous mechanisms potentially underlying diversity of FD pain phenotypes.We report on higher gene expression of calcium-activated potassium channel 3.1 (K Ca 3.1, KCNN4) and interleukin-8 by dermal fibroblasts of women with FD compared to those of men as potential contributors to sex-associated pain diversity in FD.

Study population
Between 01.09.2015 and 31.12.2018,we prospectively recruited 27 patients with FD, 13 men and 14 women, with a median age of 46 years (range 19-74) at the Fabry Center for interdisciplinary Therapy (FAZiT), University Hospital of Wu ¨rzburg, Germany.All patients underwent neurological examination, nerve conduction studies, and quantitative sensory testing (QST).We included patients, if FD was genetically confirmed and if patients showed �2 of the following three symptoms or signs: FD-associated pain (in childhood and/or adulthood), and/or paradoxical heat sensation during QST indicating small nerve fiber impairment, and/or reduced intraepidermal nerve fiber density (IENFD) in distal and/or proximal skin punch biopsies.
Controls were recruited at the Department of Neurology, University Hospital of Wu ¨rzburg from among patients'healthy (i.e.no GLA variant) family members and friends.We enrolled 20 controls (14 women, six men) with a median age of 49 years (range 22-66).Our study was approved by the Wu ¨rzburg Medical School Ethics Committee (#135/15) and participants were enrolled upon oral and written informed consent.

Skin punch biopsy
Six-mm skin punch biopsies were obtained as described previously [9].For histological determination of the IENFD and for cultivation of dermal and epidermal cells, skin samples were divided into two pieces and processed as follows.
IENFD determination.One half of the skin sample was used to histologically determine IENFD as described previously [9].Briefly, skin was fixed in 4% buffered paraformaldehyde (PFA; pH 7.4), embedded in Tissue Tek1 (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands), and stored at -80˚C until further processing.Samples were immunoreacted with antibodies against the neuronal marker protein-gene product 9.5 (PGP 9.5; 1:1000, Zytomed, Berlin, Germany) with goat anti-rabbit IgG labeled with fluorescent cyanine 3.18 (1:100; Dianova, Hamburg, Germany), for visualization of 40-μm cryosections using a Zeiss Axiophot 2 microscope (Axiophot2, Zeiss, Oberkochen, Germany) with a CCD camera (Visitron Systems, Tuchheim, Germany) and SPOT advanced software (Windows version 4.5, Diagnostic Instruments, Inc., Sterling Heights, MI, USA).We analyzed three biopsy sections per study participant each in a blinded manner.Intraepidermal nerve fibers were quantified according to published rules [21] and the mean intraepidermal nerve fiber density (i.e. the number of fibers/mm) was determined by counting intraepidermal nerve fibers, if they crossed or originated at the dermal-epidermal border.The number of fibers was divided by the length of the respective epidermis.Secondary branches and fiber fragments were spared.IENFD data were compared with our laboratory's normative data base grounding on 180 healthy controls (124 women; median age: 50 years, 20-84; 56 men; median age 53 years, 22-76) in skin biopsies from the lower leg (women: n = 109; men: n = 46) and the upper thigh (women: n = 102; men: n = 31).All skin biopsies were processed and assessed in our laboratory.
Human skin cell culture.Skin fibroblast cultures were prepared from the second half of the 6-mm skin punch biopsy as described earlier [22].In brief, epidermis and dermis were separated mechanically and placed in cell culture medium until cells started to migrate into T25 culture flasks.For human dermal fibroblast (hDF) cultivation, complete Dulbecco's Modified Eagle Medium was used: Nutrient Mixture F-12, supplemented with penicillin/streptomycin (1%) and fetal calf serum (10%) (DMEM/F-12, Life Technologies, Carlsbad, CA, USA).Human epidermal keratinocytes (hEK) were cultivated in EpiLife medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with (1%) penicillin/streptomycin and 1X Epi-Life Defined Growth Supplement (Life Technologies, Carlsbad, CA, USA).Cells were grown at 37˚C, 5% CO 2 (v/v) under regular control and medium change twice a week.For passaging, fibroblasts were incubated (1 min at 37˚C, 5% CO 2 (v/v)) with TrypLE Express (Life Technologies, Carlsbad, CA, USA) once 90% confluence was reached.Cells were stored in liquid nitrogen before further processing and were thawed and cultivated in 6-well plates, or T25 culture flasks until 80% confluence was reached.Afterwards, cells were passaged as described earlier [22].We used maximum two passages after thawing.
Immunoreaction against membrane-bound Gb3 was performed on cultured hEK cells.For this, culture medium was removed and cells were rinsed with PBS.Cells were incubated with Stx custom conjugated with Alexa Fluor 647 (1:300, Sigma-Aldrich, St. Louis, MS, USA) for 30 min on ice, afterwards washed with PBS (Merck Millipore, Billerica, MA, USA), and fixed with 4% PFA/PBS (Merck Millipore, Billerica, MA, USA) for 15 min at room temperature.After the fixation step, cells were washed three times with PBS for five minutes, counterstained with DAPI, and mounted with Aqua-Poly/Mount (Polysciences, Warrington, PA, USA).

Gene expression analysis
mRNA preparation.For fibroblast detachment and lysis, QIAzol Lysis Reagent1 (Qiagen, Hilden, Germany) was used.Applying miRNeasy Mini Kit (Qiagen, Hilden, Germany) and a Centrifuge 5417R (Eppendorf, Hamburg, Germany), we extracted mRNA from lysed cells including DNAse treatment.mRNA samples were stored at -80˚C until further processing.We used the NanoDrop™ One (Thermo Fisher Scientific, Waltham, MA, USA) for assessment of RNA quality and quantity.

Generation of induced pluripotent stem cell (iPSC) derived neurons
Sensory-like neurons of two FD patients, one with pain (FD-1) and one without pain (FD-2), and a healthy male control (Ctrl) were generated and characterized as described elsewhere [25].In brief, iPSC were generated from hDF using the StemRNA 3rd Gen reprogramming kit (Reprocell, Beltsville, MD, USA) following the manufacturer's instructions.Sensory-like neurons were cultivated according to a published protocol with minor modifications [26] and were matured for at least five weeks before analysis.

Patch-clamp analysis
Whole-cell patch-clamp analysis on iPSC-derived sensory-like neurons was performed on single cells.Bath solution consisted of 180 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 5 mM HEPES; the pH was adjusted to 7.4 [27].Borosilicate glass capillaries were used to produce patch pipettes (Kimble Chase Life Science and Research Products, Meiningen, Germany).Pipettes were heat-polished to reach an input resistance of 1 to 4 MΩ (whole-cell).The pipette recording solution contained 170 mM KCl, 2 mM MgCl 2 , 1 mM EGTA, 1 mM ATP, and 5 mM HEPES.Both solutions were adjusted to 290 mOsm.An EPC10 patch-clamp amplifier (HEKA, Ludwigshafen, Germany) was used to record currents with a sampling rate of 20 kHz.Stimulation and data acquisition were controlled by the Patchmaster software package (HEKA, Lambrecht, Germany) on a windows computer, and data analysis was performed off-line with GraphPad PRISM Version 8.01 (GraphPad Software, Inc., La Jolla, CA, USA).
iPSC-derived sensory-like neurons of a FD patient (male, 28 years, reporting FD pain attacks), and a healthy Ctrl (male, 60 years, no pain) were incubated with IL-8 (100 ng/ml, Peprotech, Hamburg, Germany) or hDF supernatant (1:1 iPSC neuron culture medium and hDF medium) for 1h at 37˚C.HDF supernatant was collected 24h after replacement of hDF medium with pure (serum-free) DMEM F12 from 80% confluent fibroblasts.Cells were incubated with female FD patient hDF (PatF), male FD patient hDF (PatM), both with a FD pain phenotype, and Ctrl (Ctrl) supernatant from healthy controls.Fibroblast supernatant was collected from cells of our investigated patient cohort.Supernatants were not pooled and all patient conditioned media samples were used as individual replicates.
For current clamp recordings, square current injections were applied to induce action potentials (AP; from 0 to 300 pA in 10 pA steps).For calculation of AP properties, the measurement function of the Patchmaster software package (HEKA, Lambrecht, Germany) was used and values of the first evoked AP were exported to an excel file for further processing and storage.We measured threshold potentials at the turning point of the overshoot and evaluated AP amplitudes as the difference between membrane potential and AP peak.AP duration was measured as the distance between depolarization and repolarization at the point of half maximum AP amplitude.The hyperpolarization amplitude was defined as the minimum potential after the AP.Rheobase was defined as minimum current to elicit the first AP.For all patch-clamp experiments and all conditions investigated, �6 cells were recorded.

Statistical analysis
SPSS 25 (IBM, Ehningen, Germany) was used for statistical analysis and GraphPad PRISM Version 8.01 (GraphPad Software, Inc., La Jolla, CA, USA) was applied for graph preparation.Kolmogorov-Smirnov test was used to test data distribution.For non-parametric gene expression analysis of skin cells, we used Kruskal-Wallis test, followed by Dunn's multiple comparison test.Normally distributed electrophysiological parameters were analyzed using two-way ANOVAs with cell line (Ctrl, FD) and treatment (naïve, Ctrl, PatF, PatM, IL-8) as independent variables.For multiple comparison, Dunnett's test was applied.P<0.05 was assumed statistically significant.

FD keratinocytes do not show intracellular, but membrane-bound Gb3
We first investigated FD and control cells for qualitative comparison.No Gb3 depositions were detected in the cytoplasm of hEK of FD patients (Fig 1A ' and 1A") and in the cytoplasm or cell membrane of control hEK (Fig 1B ,), but the immunoreaction for membrane-bound Gb3 was positive in FD hEK (Fig 1B").As expected, only few Gb3 deposits were detected in control hDF reflecting its physiological generation (Fig 1C').In contrast, dense intracellular Gb3 deposits were visible in fibroblasts of FD patients (Fig 1C").

Higher inflammatory cytokine gene expression in hDF of female FD patients than in male patients and Ctrl
We found higher IL-8 gene expression in hDF of FD patients compared to Ctrl (p<0.001,Fig 3A).Performing gene expression analysis of male and female patient subgroups, higher IL-8  expression was confirmed only for hDF of women with FD compared to hDF of men (p<0.05) and Ctrl (p<0.001,Fig 3B).There was no intergroup difference for gene expression of TNF, IL-1β, and IL-6 (Fig 3C -3E).for FD neurons incubated with Ctrl (p<0.001) or female FD hDF supernatant (p<0.001) compared to naïve Ctrl (Fig 4C ).

FD hDF supernatant leads to reduced AP thresholds
Next, we investigated rheobase currents to elicit first AP as a potential marker for pain in female FD patients.Current threshold for first AP induction was reduced in Ctrl neurons after incubation with male and female FD hDF supernatant compared to naïve neurons each).In FD neurons, incubation with female hDF supernatant and IL-8 led to reduced rheobase currents compared to naïve neurons (p<0.05each; Fig 4D).

Discussion
We have investigated Gb3-dependent cutaneous mechanisms potentially underlying sex-associated diversity in FD pain.We found higher gene expression of KCNN4 and IL-8 in dermal fibroblasts of women with FD compared to those of men and Ctrl.Additionally, we showed reduction of rheobase currents needed to elicit AP following incubation of iPSC-derived sensory-like neurons with hDF medium of female FD patients or IL-8.These results indicate a potential link between Gb3-dependent ion channel alterations and cutaneous cytokine expression influencing DRG neurons, which may contribute to pain in female FD patients.
FD-associated pain and its underlying mechanisms are incompletely understood.Although Gb3 accumulations in DRG neurons of FD patients and FD animal models [5,6] point towards a direct link of neuronal sphingolipid deposits and FD-associated pain, other mechanisms cannot be ruled out.This is particularly true for potential Gb3-independent mechanisms which were discovered recently in a zebrafish model of FD [28].We investigated skin cells, since they are known to be involved in the processing of chemical and thermal stimuli and cutaneous nociception [10,11,13].Surprisingly, we found no Gb3 deposits in the cytoplasm of FD keratinocytes, but in the cell membrane.This result indicates low production of Gb3 which, however, might eventually accumulate in the membrane due to higher turnover time.In support of this notion, a recent publication showed that intracellular deposits can be cleared by enzyme replacement while membrane-bound Gb3 remained unaffected [29].Further, although Shiga toxin labeling represents a reliable tool to visualize intracellular Gb3 accumulation, accessibility of membranous glycosphingolipids to toxin binding was shown to depend on cholesterol levels [30].The mere presence of Shiga toxin signal in the membrane of FD keratinocytes does hence not necessarily implicate missing Gb3 in the healthy controls but could arise from an overall FD-related lipid imbalance.Since comparison between fibroblasts and keratinocytes was not possible in our study due to lack of quantification and diversity in cellular labeling, we focused on the investigation of FD fibroblasts compared to control fibroblasts.
Neuronal ion channels, such as Nav 1.7 or TRPV1 are known to play a key role in small fiber-associated pain syndromes [31,32] and tactile allodynia [33].We therefore performed gene expression analysis of pain associated ion channels and found a downregulation of TRPV1 and SCN9A expression, while KCNN4 was upregulated in both FD patient groups.Our study design does not allow a functional interpretation of our data.However, in analogy to our previous findings [9], we speculate that alterations in ion channel expression in fibroblasts may be associated with dysregulation of pro-and anti-inflammatory cytokine release, which then may act on skin nociceptors.KCNN4 encoding the SK/IK channel KCa3.1 is associated with modulation of noxious chemical stimuli and tactile allodynia.This calcium-activated potassium channel is also linked with increased cytokine expression in synovial fibroblasts of rheumatoid arthritis patients [34].KCa3.1 channel inhibition led to reduced secretion of IL-6 and IL-8 [34].Cytokines are well known for contributing to neuronal hyperexcitability [35] and decreased AP thresholds [36], which may contribute to neuropathic pain.Further, we recently provided evidence for pro-inflammatory cytokine release from skin fibroblasts of patients with small fiber neuropathy [37].Specifically, we showed that keratinocytes and fibroblasts contributed differentially to nociceptor degeneration and sensitization in idiopathic small fiber neuropathy and that it was the fibroblasts that may contribute to the latter by increase in IL-6 and IL-8 expression.Given that pain FD is based on hereditary small fiber neuropathy, our current data add to the evidence supporting the active role of skin cells during the generation and maintenance of pain.Here, only IL-8 gene expression of female FD fibroblasts was upregulated compared to male FD and control fibroblasts.Therefore, we incubated iPSC-derived sensory-like neurons of a healthy control and a male FD patient with IL-8 or supernatant of Ctrl female FD or male FD fibroblasts.Incubation experiments revealed reduced rheobase in control neurons for both, supernatant from male and female fibroblasts compared to naïve neurons.However, in sensory-like neurons generated from FD patients, rheobase was decreased after incubation with supernatant only from female FD fibroblasts and with IL-8.
Reduction of AP rheobase is linked with increased neuronal excitability, potentially contributing to neuropathic pain [38,39].Male FD fibroblast supernatant failed to reduce rheobase currents in FD iPSC-derived sensory-like neurons, indicating a sex-associated difference in cytokine composition released by dermal fibroblasts.This result may provide evidence for different underlying pain mechanisms and pain phenotypes of FD patient subpopulations.Based on these findings, we propose a potential mechanism underlying sex-associated pain phenotype as illustrated in  malfunction may lead to Gb3 accumulation in FD fibroblasts.Gb3 depositions may induce increased gene expression of the specific, cytokine-linked ion channel KCa3.1 leading to higher expression of IL-8 only in female FD patients with a pain phenotype.Elevated IL-8 secretion in the vicinity of nociceptor free nerve endings of DRG neurons may then lead to sensitization via reduction of AP induction thresholds contributing to FD-associated pain in women.In men with FD and increased KCNN4 expression, other cytokines may be at play which needs further exploration.
As for the differences in expression of KCNN4 and IL8 in women and men with FD, we can only speculate that e.g. the individual genetic variant and / or genetic mosaicism in women with FD may have contributed.These factors may also be involved in the diversity of data obtained in the female cohort itself.Further, hormonal influences on skin cells and the secretion of hormones by skin cells may be diverse and together with the genetic background contribute to diversity in ion channel expression and / or cytokine profiles.
Our study has some limitations.Due to the X-linked inheritance, we could not investigate female iPSC-derived sensory-like neurons since iPSC may undergo in vitro restitution by skewed X-chromosomal inactivation [25,40], but had to restrict our experiments to sensorylike neurons obtained from male patients and controls.Further, we limited our experiments to gene expression and cannot rule out that other cytokines or proteins released by skin cells play a role in FD pain.Another limitation of our study is the small sample size, which is due to the non-high-throughput nature of our key experimental methods, as well as the heterogeneity in FD patients.Further the recently discovered fact that Gb3-independent processes may be well at play in the pathophysiology of FD [28] needs to be considered when interpreting our data.
Despite these limitations, our findings provide first insights into potential differences underlying sex-dependent diversity in FD pain phenotypes.A fully human in vitro model system was used to investigate the interaction of skin cells and sensory-like neurons in FD-associated pain.Extensive molecular and electrophysiological investigation provided evidence for a potential role of FD-induced skin cell ion channel expression alterations, leading to sensorylike neuron sensitization by cytokine secretion in female FD patients.
Due to the lack of intracellular Gb3 deposits in hEK, we continued our experiments with hDF.Gene expression analysis of hDF revealed less TRPV1 and SCN9A expression in FD hDF compared to Ctrl (p<0.001,Fig 2A', p<0.01;Fig 2A"), while there was no intergroup difference for HCN2 and KCNMA1 expression (Fig 2A"' and 2A"").KCNN4 gene expression was higher in hDF of FD patients compared to Ctrl (p<0.001,Fig 2B).When stratifying data for sex, KCNN4 gene expression was higher in men (p<0.05) and women with FD (p<0.001) compared to Ctrl (Fig 2C).SCN10A and TRPM8 were not expressed (Ct values >33 each).

Fig 5 :
mutations in the GLA gene and the consecutive enzyme

Fig 5 .
Fig 5. Potential mechanism underlying sex-associated pain phenotype.Mutation of the GLA gene leads to a non-or dys-functional enzyme, which results in Gb3 accumulation in FD fibroblasts.These deposits may be involved in increased gene expression of the cytokine-linked ion channel KCa3.1.Upregulated ion channel gene expression then may lead to an elevated expression of IL-8 only in female FD patients with a pain phenotype.Potentially increased IL-8 secretion close to nociceptor endings in the skin may then lead to sensitization via reduction of AP induction thresholds contributing to FD-associated pain.Abbreviations: GLA = α-galactosidase A; AP = action potential; FD = Fabry disease; Gb3 = globotriaosylceramide; IL-8 = interleukin 8; KCa3.1 = potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4. https://doi.org/10.1371/journal.pone.0300687.g005